The present invention relates to the field of Minor Histocompatibility Antigen typing. Bone marrow transplantation (BMT), one of the areas the invention is concerned with and the area from which the present invention originates, finds its application in the treatment of for instance severe aplastic anaemia, leukaemia and immune deficiency diseases.
In the early days of this technique may transplants failed through rejection of the graft by the host. Transplants that did succeed, however often led to an immune response by lymphocytes present in the graft against various tissues of the host (Graft versus Host Disease (GvHD)). It is now known that the GvHD response is mainly due to the presence of major histocompatibility (H) antigens which present a transplantation barrier. Therefore it is now routine practice to graft only HLA-matched materials (either from siblings or unrelated individuals) resulting in a much improved rate of success in bone marrow transplantation. However, despite this improvement, as well as improvements in pre-transplantation chemotherapy or radiotherapy and the availability of potent immunosuppressive drugs, about 20-70% of the treated patients still suffer from GvHD (the percentage is age and bone marrow donor dependent). To avoid GvHD it has been suggested to remove the cells (mature T cells) causing said reaction from the graft. This however often leads to graft failure or to recurrence of the original diseases. The cells responsible for GvHD are also the cells which often react against the original aberrant cells in for insurance leukaemia (Graft versus Leukaemia response).
Since BMT is nowadays mainly carried out with HLA matched grafts, the GvHD which still occurs must be caused by another group of antigens. It is very likely that the group of so called minor H antigens (mHag), which are non-MHC encoded histocompatibility antigens (unlike the major H antigens) are at least partially responsible for the remaining incidence of GvHD. mHag""s have originally been discovered in congeneic strains of mice in tumor rejection and skin rejection studies. In mice, the use of inbred strains has shown that mHag are encoded by almost 50 different allelically polymorphic loci scattered throughout the genome. In humans, although cumbersome to identify, mHag have been shown to exist, but their overall number and complexity remains uncertain. Minor H antigens are most likely quite different from each other and quite different from major H antigens, they are probably a diverse and elusive group of fragments of molecules which are participating in various cellular housekeeping functions. Their antigenicity may come very incidentally, as naturally processed fragments of polymorphic proteins that associate with MHC products. Some of the mH antigens appear to be widely expressed on various tissues throughout the body whereas other show limited tissue distribution.
One of the better known minor histocompatibility antigens in the H-Y antigen. H-Y is an mH antigen that can lead to rejection of HLA-matched male organ and bone marrow grafts by female recipients, and to a higher incidence of GvHD in female-to-male grafts, particularly if the female donor had been previously pregnant. The H-Y antigen may also play a role in spermatogenesis. The human H-Y antigen in an 11 residue peptide derived from SMCY, an evolutionary conserved Y chromosomal protein. Another well known mH antigen that can lead to GvHD is the HA-2 antigen. The human HA-2 antigen is an 9 residue peptide likely derived from a class 1 myosin. However, the nature of the HA-1 antigen, responsible for a majority of current cases of GvHD has remained elusive sofar. Human bone marrow transplants performed as therapeutical treatment of severe aplastic anemia, leukaemia and immune deficiency disease became available in the seventies. For the present, the long-term results of allogeneic bone marrow transplantation (BMT) have greatly improved due to the use of HLA-matched siblings as marrow donors, advanced pretransplant chemoradiotherapy, the use of potent immunsuppressive drugs as Graft-versus-Host-Disease (GVD) prophylaxis, better antibiotics and isolation procedures. Nonetheless, the results of clinical BMT reveal that the selection of MHC identical donors/recipients is not a guarantee of avoidance of GVHD or disease free survival even when donor and recipient are closely related. Allogeneic BMT especially in adults results, depending on the amount of T cell depletion of the graft, in uptil 80% of the cases in GVHD. In the HLA genotypically identical situation it amounts to 15-35% whereas in the phenotypical HLA matched recipient/donor combinations, the occurrence of GVHD is significantly higher i.e. 50-80%. Disparities for minor Histocompatibility antigens (mHag) between donor and recipient constitute a potential risk for GVHD or graft failure, which necessitate life long pharmacologic immunosuppression of organ and bone marrow transplant recipients. It is also believed that mHag are involved in the xe2x80x9cbeneficialxe2x80x9d side effect of GVHD, i.e. the Graft-versus-Leukemia activity. Several reports demonstrated the presence of anti-host mHag specific CTL in patients suffering from GVHD after HLA genotypically identical BMT. In our laboratory, much effort was put into the further characterization of a (small) number of anti-host mHag specific CTLS. Hereto, CTL clones specific for host mHag were isolated from the peripheral blood (PBL) of patients suffering from severe GvHD. mHag HA-1 specific CD8+ CTL clones were originally obtained after restimulation of in vivo primed PBLs from three patients suffering from GvHD after HLA identical but mHag nonidentical BMT. The post BMT CTL lines were cloned by limiting dilution, resulting in the isolation of a large number of mHag-specific CTL clones. Subsequent immunogeneic analyses revealed that the CTL clones (as described above) identified five non-sexlinked mHag, designated HA-1, -2, -3, -4, -5, which are recognized in an classical MHC restricted fashion. mHag HA-3 is recognized in the presence of HLA-A1 and mHag HA-1, -2, -4 and 5 require the presence of HLA-A2. Segregation studies demonstrated that each of mHag HA-1 to HA-5 is the product of a single gene segregating in a Menedelian fashion and that HA-1 and HA-2 are not coded within the HLA region. The mHag differ from each other in phenotype frequencies: mHag HA-1 appeared relatively frequent (i.e. 69%) whereas mHag HA-2 appeared very frequent (i.e. 95%) in the HLA-A2 positive healthy population. An inventory in five patients of mHag HA-1, -2, -3, -4 and -5 specific anti-host CTL response after BMT demonstrated in 3 patients clones specific for the mHag HA-1. This observation points towards the immunodominant behaviour of mhag HA-1. With regard to the mHag expressed on different tissues, we observed ubiquitous versus restricted tissue distribution of the mHag analysed. The expression of the mHag HA-1 is restricted to the cells of the haematopoietic cell lineage, such as thymocytes, peripheral blood lymphocytes, B cells, monocytes. Also the bone marrow derived professional antigen presenting cells: the dendritic cells and the epidermal Langerhans calls express the mHag HA-1. The mHag HA-1 is also expressed on clonogenic leukemic precursor cells as well as on freshly isolated myeloid and lymphoid leukemic cells, indicating that mHag specific CTLs are capable of HLA class I restricted antigen specific lysis of leukemic cells. To substantiate the importance of the human mH antigenic systems, we investigated whether the mHag are conserved in evolution between human and non human primates. Hereto, cells from non human primates were transfected with the human HLAA2.1 gene. Subsequent analyses with our human allo HLA-A2.1 and four mhag A2.1 restricted CTL clones revealed the presentation of ape and monkey allo and mHag HY. HA-1 and HA-2 peptides in the context of the transfected human HLA-A2.1 molecule by ape and monkey target cells. This implicates that the HA-1 peptide is conserved for at least 35 million years. A prospective study was performed in order to document the effect and clinical relevance of mHag in HLA genotypically identical BMT on the occurrence of acute (gradexe2x89xa72) GVHD. The results of the mHag typing using the CTL clones specific for five well defined mHag HA-1 to HA-5 demonstrated a significant correlation between mHag HA-1, -2, -4 and -5 mismatch and GVHD. A significant correlation (P=0.024) with the development of GVHD was observed when analysed for only mHag HA-1. To anlayse a putative peptide nature of the mHag HA-1, we analysed the requirement of the MHC encoded TAP1 and TAP2 gene products for mhag peptide presentation on the cell surface. The transporter genes TAP1 and TAP2 associated with antigen presentation are required for delivery of peptides from the cytosol with the endoplasmic reticulum. The availability of a human celline xe2x80x9cT2xe2x80x9d lacking both transport and proteasome subunit genes enabled us to study the processing and presentation of human mHag. We demonstrated that the (rat) transport gene products TAP1 and TAP2u were required for processing and presentation of antigenic peptides from the intracellular mH protein HA-1. Information on the TCR repertoire post BMT in man is extremely scarce. We have anlaysed the composition of the T cell receptor (TCR) V region of mHag HA-1 specific CD8+ CTL clones of DNA sequencing of the xcex1 and xcex2 chains. We observed by analyzing TCR usage of 12 clones derived from 3 unrelated individuals that the TcRxcex2 chains all used the TCRxcex2V6S9 gene segment and showed remarkable similarities within the N-D-N regions.
However, until the present invention no one has succeeded in identifying amino acid sequences of antigenic peptides relevant to the mHag HA-1 antigen, nor has anyone succeeded in the identification of the proteins from which this antigen is derived.
It is therefore an aim of the present invention to derive the amino acid sequence of the HA-1 antigen.
It is also an aim of the present invention to derive the nucleic acid sequence of the HA-1 antigen, more particularly the cDNA and the genomic sequences encoding HA-1 antigens.
It is also an aim of the present invention to provide primers and probes enabling typing of HA-1 antigens.
It is also an aim of the present invention to provide kits allowing to type HA-1 antigens.
The present inventors have now for the first time identified a peptide which is a relevant part of mHag HA-1. The present inventors have also identified the cDNA sequence as well as the genomic sequence of two HA-1 alleles.
The present inventors describe for the first time a (poly)peptide comprising a T-cell epitope obtainable from the minor Histocompatibility antigen HA-1 comprising the sequence VLXDDLLEA (SEQ ID NO: 17) or a derivative thereof having similar functional or immunological properties, wherein X represents a histidine (H) or an arginine (R) residue.
Diagnostic applications envisaged in this invention include, but are not limited to HA-1 typing, detection of genetic aberrances and the like.
On the basis of the peptide described herein genetic probes or primers were produced which can be used to screen for the gene encoding the protein. On the basis of the peptide described herein anti-idiotypic B cells and/or T cells and antibodies can be produced. Various techniques, to allow detection of suitable donors or recipients, may be used, based on amplification of HA-1 related nucleic acid sequences or on the immunological detection of HA-1 related peptide sequences as set out further.
According to one embodiment, the present invention relates to a method for typing of alleles of the Minor Histocompatibility Antigen HA-1 in a sample comprising the detection of polymorphive nucleotides is the cDNA or genomic nucleic acids of said alleles, more particularly the H and R alleles of HA-1 as set out in FIG. 5.
In a preferred embodiment said method of typing will be a method of genomic DNA typing. Alternatively said method may also be a method of cDNA typing.
Another embodiment of the present invention relates to genomic typing of alleles of the Minor Histocompatibility Antigen HA-1 in a sample, with said method comprising:
a) contacting the genomic polynucleic acids in the sample with at least one pair of primers, whereby the 5xe2x80x2- and/or the 3xe2x80x2-primer of said at least one pair of primers specifically hybridize to target regions comprising polymorphic nucleotides in said alleles, and performing an amplification reaction;
b) for each of said at least one pair of primers detecting whether or not in step a) an amplification product is formed;
c) inferring from the result of step b) which HA-1 allele is present in said sample.
According to a preferred embodiment, the present invention relates to a method as described above, further characterized in that said alleles of the Minor Histocompatibility Antigen HA-1 are the H allele and the R allele as shown in FIG. 5.
The present invention teaches that, unexpectedly, the primers used in the RT-PCR method, do not lead to amplification of a specific polynucleic acid fragment when genomic DNA is used as a template. To solve this problem, the present invention also discloses the genomic structure of the HA-1 locus. As explained in Example 3, analysis of the genomic structure shows that the HA-1 peptide is encoded by two exons (FIG. 5). A splice donor site is located four nucleotides after the polymorphic codon in the HA-1 coding sequence. Therefore, setting up a method for genomic typing of the HA-1 antigen requires sequence information of the intron interrupting the HA-1 codon sequence. This sequence information is provided by the present invention and is shown below as SEQ ID NO 1.
gtg aga gcc acg ggg aca ccg agg cct ggg tgg aag aca gag cca gac cca agg gag gat gga ggg agg gac ttg ggg agg ctc aga agg gag gga ggc tca gat ggc agg gag ggc tgt gtg gaa gag gcc atg aca gct aag gct ctg agg gat gtg tag gag ttt ggt ggg gga gtc cct gag cgt aca ctg gct caa gag ggt gco cac ttt att ttt rtt aaa gga tct gtt ggc aat tag gag gga aag gca gag gat atg tcc cat gca cag gct cag aaa cac gaa aac aga gac tgc att tgg ggg cca agg tgt ggg gtg ccg ctg gtg tag gat gan ggc atg aca acg cca ggc aga agg goa at SEQ ID NO 1
This sequence represents part of the interrupting intron (indicated as intron a in FIG. 5), the first nucleotide of this sequence being the first nucleotide of intron a. The present invention thus also relates to an isolated polynucleic acid identified by SEQ ID NO 1, or an isolated polynucleic acid displaying at least 80%, or at least 90%, or at least 95%, or at least 99% sequence homology to SEQ ID NO 1, or any fragment of said polynucleic acids that can be used as a primer or as a probe.
Sequence information corresponding to another part of intron a, more particularly the part which is situated in front of exon b (FIG. 5) has been dissolved in the EMBL database under accession number AC004151. However, this sequence is not suitable for the design of primers for the above-mentioned method, since the length of the amplified fragment would lower the efficiency of the amplification reaction.
The present invention also relates to isolated polynucleic acid identified by SEQ ID NO 17 (HA-1 R allele), or an isolated polynucleic acid displaying at least 80%, or at least 90%, or at least 95%, or at least 99% sequence homology to SEQ ID NO 17, or any fragment of said polynucleic acid that can be used as a primer or as a probe.
The present invention also relates to isolated polynucleic acid identified by SEQ ID NO 18 (HA-1 R allele), or an isolated polynucleic acid displaying at least 80%, or at least 90%, or at least 95%, or at least 99% sequence homology to SEQ ID NO 18, or any fragment of said polynucleic acid that can be used as a primer or as a probe.
The present invention also relates to any part of the sequence of KIAA0223 (GENBANK Acc. No. D 86976) which lies on the borders of SEQ ID NO 17 or 18, particularly sequence lying on the 5xe2x80x2 side of SEQ ID NO 17 or 18 in the KIA0223 sequence. Such sequences are useful for the design of primers for HA-1 typing as disclosed in the present claims.
For detection of the amplification product mentioned in step b above, different methods known in the art, may be used. One method consists of subjecting the mixture obtained after the amplification reaction to gel electrophoresis and visually detecting the amplification product after nucleic staining. Alternatively, the amplification product may be labeled, for instance by using labeled primers, and may be captured on a solid support, for instance by hybridization, and may be detected on the solid support. It is clear, however, that other detection methods are also within the scope of the present invention.
According to a more preferred embodiment, the present invention relates to a method as indicated above, further characterized in that:
said at least one pair of primers comprise a 5xe2x80x2-primer that specifically hybridizes to a target region comprising the nucleotides at position 4 or at positions 4 and 8 in the HA-1 allele, or
said at least one pair of primers comprises a 3xe2x80x2-primer that specifically hybridizes to a target region comprising the nucleotides at position 8 or at positions 4 and 8 in the HA-1 allele, with said positions being indicated in FIG. 5.
According to an even more preferred embodiment, the present invention relates to a method as indicated above, further characterized in that:
said 5xe2x80x2-primer is combined with a 3xe2x80x2-primer specifically hybridizing to a target region in introl a, and/or
said 3xe2x80x2-primer is combined with a 5xe2x80x2-primer specifically hybridizing to a target region in exon a,
with intron a and exon a being indicated in FIG. 5.
According to this embodiment, said target region in intron a is ideally located in the sequence identified by SEQ ID NO 1, as explained above. Also the target region of said 3xe2x80x2-primer that is combined with a 5xe2x80x2-primer specifically hybridizing to a target region in exon a, will necessarily overlap with the sequence identified by SEQ ID NO 1.
According to an even more preferred embodiment, the present invention relates to a method as indicated above, further characterized in that the primers are chosen from Table 1:
Set 1 consists of a common 5xe2x80x2-primer (forward) and two different 3xe2x80x2-primers (reverse), one for the H allele and one for the R allele. The target region of the common 5xe2x80x2-primer is located in exon a. The target region of the 3xe2x80x2-position comprises the polymorphic nucleotides at positions 4 and 8 in the HA-1 coding sequence (FIG. 5) and partly overlaps with the sequence identified by SEQ ID NO 1. Set 2 consists of a common 3xe2x80x2-primer and two different 5xe2x80x2-primers. The target region of the common primer is located in the sequence identified by SEQ ID NO 1, whereas the target regions of the 5xe2x80x2-primers are located in exon a and comprise the polymorphic nucleotides at position 4 and 8. Example 6 shows a genomic type experiment making use of these primer sets.
According to another preferred embodiment, the present invention relates to a diagnostic kit for genomic typing of alleles of the Minor Histocompatibility Antigen HA-1 according to any of the methods indicated above, with said kit comprising:
a) at least one primer according to any of the methods indicated above;
b) optionally, an enzyme and/or reagent enabling the amplification reaction;
c) optionally, means enabling direction of the amplified products.
According to another preferred embodiment, the present invention relates to a method for genomic typing of alleles of the Minor Histocompatibility Antigen HA-1 in a sample, with said method comprising:
a) amplifying a fragment of said alleles, with said fragment comprising at least one polymorphic nucleotide, by use of at least one pair of primers specifically hybridizing to conserved target regions in said alleles;
b) hybridizing the amplified product of step a) to at least one probe specifically hybridizing to a target region comprising one or more polymorphic nucleotides in said allele;
c) inferring from the result of step b) which HA-1 allele is present in said sample.
According to a more preferred embodiment, the present invention relates to a method as indicated above, further characterized in that said alleles of the Minor Histocompatibility Antigen HA-1 are the H allele and the R allele.
According to an even more preferred embodiment, the present invention relates to a method as indicated above, further characterized in that said at least one pair of primers comprises 5xe2x80x2-primer specifically hybridizing to a conserved target region in exon a and/or a 3xe2x80x2-primer specifically hybridizing to a conserved target region in intron a, with exon and intron a being indicated in FIG. 5.
Ideally, the target region of said 3xe2x80x2-primer is located in the sequence identified as SEQ ID NO 1. Obviously, the target region of said 3xe2x80x2-primer may also be located downstream of this sequence, i.e. in intron a, intron b, exon b, or even downstream of exon b, but the efficacy of the amplification reaction is likely to be lower as the amplified fragment becomes longer. According to an even more preferred embodiment, the present invention relates to a method as indicated above, further characterized in that said at least one probe specifically hybridizes to a target region comprising the nucleotides at position 4 and/or 8 in the HA-1 allele, with said positions being indicated in FIG. 5.
According to an even more preferred embodiment, the present invention relates to a method as indicated above, further characterized in that said primers and/or said probes are chosen from Table 2:
Primers 3P1 and 3P2 specifically hybridize target regions in SEQ ID NO 1. The target region of primer 3P3 is located downstream of exon b. The probes of Table 2 all specifically hybridize to target regions overlapping with the exon a-intron a boundary. The probes with SEQ ID NO 11 to 16 have been optimized to function in combination at the same conditions in a LiPA assay (see below). The skilled man will recognize that the probes and primers with SEQ ID NO 2 to 16 may be adapted by addition or deletion of one or more nucleotides at their extremities. Such adaptations may be required if the conditions of amplification or hybridization are changed, or if the amplified material is RNA instead of DNA, as in the case in the NASBA system. Different techniques can be applied to perform the sequence-specific hybridization methods of the present invention. These techniques may comprise immobilizing the amplified HA-1 polynucleic acids on a solid support and performing hybridization with labelled oligonucleotide probes. Genomic polynucleic acids may also be immobilized on a solid support without prior amplification and subjected to hybridization. Alternatively, the probes may be immobilized on a solid support and hybridization may be performed with labelled HA-1 polynucleic acids, preferably after amplification. This technique is called reverse hybridization. A convenient reverse hybridization technique is the line probe assay (LiPA). This assay uses oligonucleotide probes immobilized as parallel lines on a solid support strip (Stuyver et al., 1993). It is to be understood that any other technique for genomic typing of HA-1 alleles is also conveyed by the present invention.
It is clear that the present invention also relates to any of the primers with SEQ ID NO 2 to 10 and to any of the probes with SEQ ID NO 11 to 16, with said primers and said probes being for use in a method for genomic typing of alleles of the Minor Histocompatibility Antigen HA-1.
According to another preferred embodiment, the present invention relates to a diagnostic kit for genomic typing of alleles of the Minor Histocompatibility Antigen HA-1 according to any of the sequence-specific hybridization methods indicated above, with said kit comprising:
a) at least one primer according to any of the methods indicated above;
b) optionally, an enzyme and/or reagents enabling the amplification reaction, and/or reagents enabling the hybridization reaction.
According to another preferred embodiment, the present invention relates to a diagnostic kit for genomic typing of alleles of the Minor Histocompatibility Antigen HA-1 according to any of the sequence-specific hybridization methods indicated above, with said kit comprising:
a) at least one primer according to any of the methods indicated above;
b) at least one probe according to any of the methods indicated above;
c) optionally, an enzyme and/or reagents enabling the amplification reaction, and/or reagents enabling the hybridization reaction.
According to another embodiment, the present invention also relates to a method for typing of alleles of the Minor Histocompatibility Antigen HA-1 by means of sequencing said allele.
According to another embodiment, the present invention also relates to kits for performing said sequencing method.
According to another embodiment, the present invention also relates to a method for typing HLA-1 alleles comprising using antibodies specifically detecting the HA-1 alleles shown in FIG. 5. Said antibodies will be preferably monoclonal antibodies and can be produced by any method shown in the art.
According to another embodiment, the present invention also relates to a diagnostic kit for typing HLA-1 allele comprising using antibodies specifically detecting the HA-1 alleles shown in FIG. 5.
The following definitions and explanations will permit a better understanding of the present invention.
The target material in the samples to be anlaysed will be genomic DNA or amplified versions thereof. These molecules are in this application also termed xe2x80x9cpolynucleic acidsxe2x80x9d. Well-known extraction and purification procedures are available for the isolation of RNA or DNA from a sample (e.g. in Sambrook et al., 1989).
A xe2x80x9cpolymorphic nucleotidexe2x80x9d refers to a nucleotide of the sequence of a given HA-1 allele that differs from at least one of the nucleotides that are found at the corresponding position in other HA-1 alleles.
The term xe2x80x9ctypingxe2x80x9d of an HA-1 allele refers to identification of the allele, i.e. detection of the allele and discrimination of the allele from other HA-1 alleles. The term xe2x80x9cprobexe2x80x9d according to the present invention refers to a single-stranded oligonucleotide which is designed to specifically hybridize to HA-1 polynucleic acids. Preferably, the probes of the invention are about 5 to 50 nucleotides long, more preferably from about 10 to 30 nucleotides. Particularly preferred lengths of probes include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. The nucleotides as used in the present invention may be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridization characteristics.
The term xe2x80x9cprimerxe2x80x9d refers to a single stranded oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products. Preferably the primer is about 5-50 nucleotides long. Specific length and sequence will depend on the complexity of the required DNA or RNA target, as well as on the conditions at which the primer is used, such as temperature and ionic strength. It is to be understood that the primers of the present invention may be used as probes and vice versa, provided that the experimental conditions are adapted.
The expression xe2x80x9csuitable primer pairxe2x80x9d in this invention refers to a pair of primers allowing specific amplification of a HA-1 polynucleic acid fragment. The term xe2x80x9ctarget regionxe2x80x9d of a probe or a primer according to the present invention is a sequence within the HA-1 polynucleic acids to which the probe or the primer is completely complementary or partially complementary (i.e. with some degree of mismatch). It is to be understood that the complement of said target sequence is also a suitable target sequence in some cases.
xe2x80x9cSpecific hybridizationxe2x80x9d of a probe to a target region of the HA-1 polynucleic acids means that said probe forms a duplex with part of this region or with the entire region under the experimental conditions used, and that under those conditions said probe does not form a duplex with other regions of the polynucleic acids present in the sample to be anlaysed. xe2x80x9cSpecific hybridizationxe2x80x9d of a primer to a target region of the HA-1 polynucleic acids means that, during the amplification step, said primer forms a duplex with part of this region or with the entire region under the experimental conditions used, and that under those conditions said primer does not form a duplex with other regions of the polynucleic acids present in the sample to be analysed. It is to be understood that xe2x80x9cduplexxe2x80x9d as used hereby, means a duplex that will lead to specific amplification.
xe2x80x9cSpecific amplificationxe2x80x9d of a fragment of the HA-1 polynucleic acids means amplification of the fragment for which the primers were designed, and not of any other fragment of the polynucleic acids present in a sample.
The fact that amplification primers do not have to match exactly with the corresponding target sequence in the template to warrant proper amplification is amply documented in the literature (Kwok et al, 1990). However, when the primers are not completely complementary to their target sequence, it should be taken into account that the amplified fragments will have the sequence of the primers and not of the target sequence. Primers may be labelled with a label of choice (e.g. biotin). The amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu and Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Dunk, 1990) or amplification by means of QB replicase (Lomeli et al., 1989) or any other suitable method to amplify nucleic acid molecules known in the art.
Probe and primer sequences are represented throughout the specification as single stranded DNA oligonucleotides from the 5xe2x80x2 to the 3xe2x80x2 end. It is obvious to the man skilled in the art that any of the below-specified probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U).
The probes according to the invention can be prepared by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequence, if need be by excision of the latter from the cloned plasmids by use of the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight. The probes according to the present invention can also be synthesized chemically, for instance by the conventional phospho-trietter method.
The oligonucleotides used as primers or probes may also comprise nucleotide analogues such as phosphorothiates (Matsukura et al., 1987), alkylphosphorothiates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et al., 1984). As most other variations or modifications introduced into the original DNA sequences of the invention these variations will necessitate adaptations with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However the eventual results of hybridization will be essentially the same as those obtained with the unmodified oligonucleotides. The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonucleotides molecules, etc.
The term xe2x80x9csolid supportxe2x80x9d can refer to any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low. Usually the solid substrate will be a microtiter plate, a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead) or a chip. Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fixation or improve the hybridization efficiency. Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NH2 groups, SH groups, carboxylic groups, or coupling with biotin, haptens or proteins.
The term xe2x80x9clabelledxe2x80x9d refers to the use of labelled nucleic acids. Labelling may be carried out by the use of labelled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1988) or Bej et al. (1990) or labelled primers, or by any other method known to the person skilled in the art. The nature of the label may be isotopic (32P, 35S, etc.) or non-isotopic (biotin, digoxigenin, etc.).
The xe2x80x9cbiological samplexe2x80x9d may be for instance blood, mouth swab or any other sample comprising genomic DNA.
For designing probes with desired characteristics, the following useful guidelines known to the person skilled in the art can be applied.
Because the extent and specificity of hybridization reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The importance and effect of various assay conditions are explained further herein.
The stability of the [probe:target] nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long AT-rich sequences, by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and %GC result in a Tm about 2-10xc2x0 C. higher than the temperature at which the final assay will be performed. The base composition of the probe is significance because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher G-C content will be more stable at higher temperatures.
Conditions such as inionic strength and incubation temperature under which a probe will be used should also be taken into account when designing a probe. It is known that the degree of hybridization will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strength. On the other hand, chemical reagents, such as formamide, urea, DMSO and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridization. Destabilization of the hydrogen bonds or such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5xc2x0 C. below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity.
It is desirable to have probes which hybridize only under conditions of high stringency. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. The degree of stringency is chosen such as to maximize the difference in stability between the hybrid formed with the target and the non-target nucleic acid.
Regions in the target DNA or RNA which are known to form strong internal structures inhibitory to hybridization are less preferred. Likewise, probes with extensive self-complementarity should be avoided. As explained above, hybridization is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. There can be intramolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization may be greatly increased. Computer programs are available to search for this type of interraction. However, in certain instances, it may not be possible to avoid this type of interaction.
Standard hybridization and wash conditions are disclosed in the Materials and Methods section of the Examples. Other conditions are for instance 3xc3x97 SSC (Sodium Salt Citrate), 20% deionized FA (Formamide) at 50xc2x0 C. Other solutions (SSPE (Sodium saline phosphate EDTA), TMAC (Tetramethyl ammonium Chloride), etc.) and temperatures can also be used provided that the specificity and sensitivity of the probes is maintained. When needed, slight modifications of the probes in length or in sequence have to be carried out to maintain the specificity and sensitivity required under the given circumstances.
The term xe2x80x9chybridization bufferxe2x80x9d means a buffer allowing a hybridization reaction between the probes and the polynucleic acids present in the sample, or the amplified products, under the appropriate stringency conditions.
The term xe2x80x9cwash solutionxe2x80x9d means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.